Long-term Consequences on Soil Fungal Community Structure: Monoculture Planting and Natural Regeneration.

Understanding the regeneration and succession of belowground communities, particularly in forests, is vital for maintaining ecosystem health. Despite its importance, there is limited knowledge regarding how fungal communities change over time during ecosystem development, especially under different forest restoration strategies. In this study, we focused on two restoration methods used in northern Japan: monoculture planting and natural regeneration. We examined the responses of the fungal community to monoculture plantations (active tree planting) and naturally regenerated (passive regeneration) forests over a 50-year chronosequence, using natural forests as a reference. Based on DNA metabarcoding, we assessed the richness of fungal Operational Taxonomic Units (OTUs) and their dissimilarity. Our findings revealed that soil fungal richness remained stable after natural regeneration but declined in monoculture plantations, from 354 to 247 OTUs. While the compositional dissimilarity of fungal assemblages between monoculture plantations and natural forests remained consistent regardless of the time since tree planting, it significantly decreased after natural regeneration, suggesting recovery to a state close to the reference level. Notably, the composition of key functional fungal groups-saprotrophic and ectomycorrhizal- has increasingly mirrored that of natural forests over time following passive natural regeneration. In summary, our study suggests that monoculture plantations may not be effective for long-term ecosystem function and service recovery because of their limited support for soil fungal diversity. These results underscore the importance of natural regeneration in forest restoration and management strategies.

Comparison of safety and effectiveness between etanercept biosimilar LBEC0101 and reference in patients with rheumatoid arthritis in real-world data using the KURAMA cohort.

OBJECTIVES: Biosimilars are anticipated to be widely used in the treatment of rheumatoid arthritis (RA), owing to their cost efficiency; LBEC0101 was the first etanercept (ETN) biosimilar approved in Japan. However, there are limited real-world data comparing its safety and effectiveness with those of a reference product. METHODS: This study used data from the Kyoto University Rheumatoid Arthritis Management Alliance cohort, including patients with RA who received ETN therapy-ETN reference product (ETN-RP) or LBEC0101-between 2015 and 2021. Serum ETN levels were measured using liquid chromatography-tandem mass spectrometry. RESULTS: The 1-year continuation rates of ETN-RP and LBEC0101 were 58.7% and 74.4%, respectively. Effectiveness of treatment was evaluated in 18 patients; both products significantly reduced the 28-joint RA disease activity score and erythrocyte sedimentation rate (DAS28-ESR). Moreover, to determine equivalence, we analysed 11 patients who switched from ETN-RP to LBEC0101; the DAS28-ESR and serum ETN levels before and after switching were not significantly different. CONCLUSIONS: This real-world cohort study confirmed that the biosimilar of ETN, LBEC0101, was comparable to the reference product in terms of continuation rate, effectiveness at initiation of introduction, and effect persistence before and after switching in clinical practice.

Crystal structure of adenosine 5'-phosphosulfate kinase isolated from Archaeoglobus fulgidus.

The 3'-phosphoadenosine-5'-phosphosulfate (PAPS) molecule is essential during enzyme-catalyzed sulfation reactions as a sulfate donor and is an intermediate in the reduction of sulfate to sulfite in the sulfur assimilation pathway. PAPS is produced through a two-step reaction involving ATP sulfurylase and adenosine 5'-phosphosulfate (APS) kinase enzymes/domains. However, archaeal APS kinases have not yet been characterized and their mechanism of action remains unclear. Here, we first structurally characterized APS kinase from the hyperthermophilic archaeon Archaeoglobus fulgidus, (AfAPSK). We demonstrated the PAPS production activity of AfAPSK at the optimal growth temperature (83 °C). Furthermore, we determined the two crystal structures of AfAPSK: ADP complex and ATP analog adenylyl-imidodiphosphate (AMP-PNP)/Mg2+/APS complex. Structural and complementary mutational analyses revealed the catalytic and substrate recognition mechanisms of AfAPSK. This study also hints at the molecular basis behind the thermal stability of AfAPSK.

Versatile aliphatic polyester biosynthesis system for producing random and block copolymers composed of 2-, 3-, 4-, 5-, and 6-hydroxyalkanoates using the sequence-regulating polyhydroxyalkanoate synthase PhaCAR.

BACKGROUND: Polyhydroxyalkanoates (PHAs) are microbial polyesters synthesized by PHA synthases. Naturally occurring PHA copolymers possess a random monomer sequence. The development of PhaCAR, a unique sequence-regulating PHA synthase, has enabled the spontaneous biosynthesis of PHA block copolymers. PhaCAR synthesizes both a block copolymer poly(2-hydroxybutyrate)-b-poly(3-hydroxybutyrate) [P(2HB)-b-P(3HB)], and a random copolymer, poly(3HB-co-3-hydroxyhexanoate), indicating that the combination of monomers determines the monomer sequence. Therefore, in this study, we explored the substrate scope of PhaCAR and the monomer sequences of the resulting copolymers to identify the determinants of the monomer sequence. PhaCAR is a class I PHA synthase that is thought to incorporate long-main-chain hydroxyalkanoates (LMC HAs, > C3 in the main [backbone] chain). Thus, the LMC monomers, 4-hydroxy-2-methylbutyrate (4H2MB), 5-hydroxyvalerate (5HV), and 6-hydroxyhexanoate (6HHx), as well as 2HB, 3HB, and 3-hydroxypropionate (3HP) were tested. RESULTS: Recombinant Escherichia coli harboring PhaCAR, CoA transferase and CoA ligase genes was used for PHA production. The medium contained the monomer precursors, 2HB, 3HB, 3HP, 4H2MB, 5HV, and 6HHx, either individually or in combination. As a result, homopolymers were obtained only for 3HB and 3HP. Moreover, 3HB and 3HP were randomly copolymerized by PhaCAR. 3HB-based binary copolymers P(3HB-co-LMC HA)s containing up to 2.9 mol% 4H2MB, 4.8 mol% 5HV, or 1.8 mol% 6HHx were produced. Differential scanning calorimetry analysis of the copolymers indicated that P(3HB-co-LMC HA)s had a random sequence. In contrast, combining 3HP and 2HB induced the synthesis of P(3HP)-b-P(2HB). Similarly, P(2HB) segment-containing block copolymers P(3HB-co-LMC HA)-b-P(2HB)s were synthesized. Binary copolymers of LMC HAs and 2HB were not obtained, indicating that the 3HB or 3HP unit is essential to the polymer synthesis. CONCLUSION: PhaCAR possesses a wide substrate scope towards 2-, 3-, 4-, 5-, and 6-hydroxyalkanoates. 3HB or 3HP units are essential for polymer synthesis using PhaCAR. The presence of a 2HB monomer is key to synthesizing block copolymers, such as P(3HP)-b-P(2HB) and P(3HB-co-LMC HA)-b-P(2HB)s. The copolymers that did not contain 2HB units had a random sequence. This study's results provide insights into the mechanism of sequence regulation by PhaCAR and pave the way for designing PHA block copolymers.

Sodium bicarbonate regulates nitric oxide production in mouse macrophage cell lines stimulated with lipopolysaccharide and interferon γ.

Macrophages are known to play pivotal roles in host-defense through inflammation via both innate and acquired immune systems, and so on. In an earlier paper, we showed the influence of the type of culture medium, Ham's F-12 or DMEM, on activated macrophage phenotypes induced by LPS and IFNγ. The production of nitric oxide (NO), pro-inflammatory cytokines such as TNFα and IL-1ß, as well as the induction of superoxide-generating activity of J774.1/JA-4 cells was different depending on the type of culture medium. In this present study, we showed that sodium bicarbonate concentrations in these culture media, 14 mM in Ham's F-12 and 44 mM in DMEM, were crucial to explaining the differences in the induction of activated macrophage phenotypes, especially in that of iNOS. A concentration-dependent change in pH did not result in any remarkable difference in iNOS expression or NO production. Moreover, high sodium bicarbonate in culture medium increased not only NO production but also TNFα production in the activated macrophages. These results suggest that sodium bicarbonate would be a regulatory factor of NO and TNFα production in macrophages and that its concentration has a crucial role in macrophage activation.

An evaluation and implementation of rule-based Home Energy Management System using the Rete algorithm.

In recent years, sensors become popular and Home Energy Management System (HEMS) takes an important role in saving energy without decrease in QoL (Quality of Life). Currently, many rule-based HEMSs have been proposed and almost all of them assume "IF-THEN" rules. The Rete algorithm is a typical pattern matching algorithm for IF-THEN rules. Currently, we have proposed a rule-based Home Energy Management System (HEMS) using the Rete algorithm. In the proposed system, rules for managing energy are processed by smart taps in network, and the loads for processing rules and collecting data are distributed to smart taps. In addition, the number of processes and collecting data are reduced by processing rules based on the Rete algorithm. In this paper, we evaluated the proposed system by simulation. In the simulation environment, rules are processed by a smart tap that relates to the action part of each rule. In addition, we implemented the proposed system as HEMS using smart taps.

Bicarbonate enhances the inflammatory response by activating JAK/STAT signalling in LPS + IFN-γ-stimulated macrophages.

Macrophages, which develop by changing their functions according to various environmental conditions and stimuli, defend against the pathogens and play roles in homoeostasis and disease states. Bicarbonate (HCO3-) is important in the maintenance of intracellular and extracellular pH in the body. However, the effects of bicarbonate on macrophage function have not been examined. In this study, we investigated the effects of bicarbonate on macrophage activation in lipopolysaccharide (LPS) and interferon (IFN)-γ (LPS + IFN-γ)-stimulated murine macrophage-like RAW264.7 cells. The expression of the interleukin (IL)-6, inducible nitric oxide (NO) synthase and cyclooxygenase-2 genes was enhanced by sodium bicarbonate (NaHCO3) in a concentration-dependent manner in LPS + IFN-γ-stimulated RAW264.7 cells. The production of IL-6, NO2- and prostaglandin E2 was also increased by treatment with NaHCO3 in these cells. Moreover, NaHCO3-mediated elevation of inflammatory gene expression was abrogated by solute carrier (SLC) transporter inhibitors. Furthermore, its NaHCO3-mediated activation was negated by a JAK inhibitor , tofacitinib. NaHCO3-enhanced phosphorylation of STAT1, and its enhancement was abrogated by pre-treating with SLC transporter inhibitors in LPS + IFN-γ-stimulated RAW264.7 cells. In addition, similar results were obtained in murine bone marrow-derived macrophages. These results indicate that bicarbonate enhanced the inflammatory response through the JAK/STAT signalling in LPS + IFN-γ-stimulated macrophages.

Induction of different activated phenotypes of mouse peritoneal macrophages grown in different tissue culture media.

The role of activated macrophages in the host defense against pathogens or tumor cells has been investigated extensively. Many researchers have been using various culture media in in vitro experiments using macrophages. We previously reported that J774.1/JA-4 macrophage-like cells showed great differences in their activated macrophage phenotypes, such as production of reactive oxygen, nitric oxide (NO) or cytokines depending on the culture medium used, either F-12 (Ham's F-12 nutrient mixture) or Dulbecco modified Eagle's medium (DMEM). To examine whether a difference in the culture medium would influence the functions of primary macrophages, we used BALB/c mouse peritoneal macrophages in this study. Among the activated macrophage phenotypes, the expression of inducible NO synthase in LPS- and/or IFN-γ-treated peritoneal macrophages showed the most remarkable differences between F-12 and DMEM; i.e., NO production by LPS- and/or IFN-γ-treated cells was far lower in DMEM than in F-12. Similar results were obtained with C57BL mouse peritoneal macrophages. Besides, dilution of F-12 medium with saline resulted in a slight decrease in NO production, whereas that of DMEM with saline resulted in a significant increase, suggesting the possibility that DMEM contained some inhibitory factor(s) for NO production. However, such a difference in NO production was not observed when macrophage-like cell lines were examined. These results suggest that phenotypes of primary macrophages could be changed significantly with respect to host inflammatory responses by the surrounding environment including nutritional factors and that these altered macrophage phenotypes might influence the biological host defense.

Influence of the culture medium on the production of nitric oxide and expression of inducible nitric oxide synthase by activated macrophages in vitro.

Macrophages play an important role in immune and inflammatory responses, and have been extensively studied in vitro using culture media such as RPMI1640 medium, Dulbecco's modified Eagle medium (DMEM), and Ham's F-12 medium (F-12). We found that the activation phenotypes of a murine macrophage-like cell line, J774.1/JA-4, were obviously different in two distinct culture media (F-12 and DMEM), both of which were supplemented with 10% of the same fetal bovine serum (FBS). Among these phenotypes, nitric oxide (NO) production as well as inducible NO synthase (iNOS) expression, induced by lipopolysaccharide (LPS) and interferon-γ (IFN-γ), were remarkably different. iNOS expression was higher in the macrophages cultured in DMEM than in F-12 for 20 h, while no significant differences were shown in NO production between in F-12 and DMEM. It might be the reason why DMEM have reduced NO production by the induced iNOS. Besides, [Formula: see text]-generating activity, and production of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in the activated macrophages were also different between the cultures in F-12 and DMEM. These results suggest that F-12 and DMEM contain certain components responsible for modification of macrophage activation processes and/or macrophage functions. Our present results provide evidence that the choice of culture medium is important in the study and analysis of macrophage activation.